Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity.

2.50
Hdl Handle:
http://hdl.handle.net/10033/620812
Title:
Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity.
Authors:
Zielinski, Marco; Kahl, Silke; Standfuss-Gabisch, Christine; Cámara, Beatriz; Seeger, Michael; Hofer, Bernd ( 0000-0002-5174-4021 )
Abstract:
Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity. 2006, 72 (3):2191-9 Appl. Environ. Microbiol.
Journal:
Applied and environmental microbiology
Issue Date:
Mar-2006
URI:
http://hdl.handle.net/10033/620812
DOI:
10.1128/AEM.72.3.2191-2199.2006
PubMed ID:
16517671
Type:
Article
Language:
en
ISSN:
0099-2240
Appears in Collections:
Publications of the research group Chemical Biology (CBIO)

Full metadata record

DC FieldValue Language
dc.contributor.authorZielinski, Marcoen
dc.contributor.authorKahl, Silkeen
dc.contributor.authorStandfuss-Gabisch, Christineen
dc.contributor.authorCámara, Beatrizen
dc.contributor.authorSeeger, Michaelen
dc.contributor.authorHofer, Bernden
dc.date.accessioned2017-02-08T10:11:02Z-
dc.date.available2017-02-08T10:11:02Z-
dc.date.issued2006-03-
dc.identifier.citationGeneration of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity. 2006, 72 (3):2191-9 Appl. Environ. Microbiol.en
dc.identifier.issn0099-2240-
dc.identifier.pmid16517671-
dc.identifier.doi10.1128/AEM.72.3.2191-2199.2006-
dc.identifier.urihttp://hdl.handle.net/10033/620812-
dc.description.abstractAryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subject.meshAmino Acid Sequenceen
dc.subject.meshAmino Acid Substitutionen
dc.subject.meshBiphenyl Compoundsen
dc.subject.meshBurkholderiaen
dc.subject.meshDioxygenasesen
dc.subject.meshDirected Molecular Evolutionen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshMutagenesisen
dc.subject.meshSubstrate Specificityen
dc.titleGeneration of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalApplied and environmental microbiologyen
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